南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (10): 1725-1733.doi: 10.12122/j.issn.1673-4254.2023.10.10

• • 上一篇    下一篇

槲皮素诱导肝星状细胞凋亡：基于调控miR-146影响PI3K/Akt信号通路

高晓阳，赵晓璐，张春艳，颜羽昕，金 蓉，马月宏   

  1. 内蒙古医科大学基础医学院，内蒙古自治区 呼和浩特 010000
  • 出版日期:2023-10-20 发布日期:2023-11-02

Quercetin induces hepatic stellate cell apoptosis by inhibiting the PI3K/Akt signaling pathway via upregulating miR-146

GAO Xiaoyang, ZHAO Xiaolu, ZHANG Chunyan, YAN Yuxin, JIN Rong, MA Yuehong   

  1. Department of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot 010000, China
  • Online:2023-10-20 Published:2023-11-02

RichHTML

23

PDF

270

摘要： 目的 探讨槲皮素（Que）通过调控肝星状细胞内miR-146水平影响PI3K/Akt信号通路诱导细胞凋亡的作用机制。方法 以大鼠肝星状细胞系（HSC-T6）为研究对象，设计实验分组：空白对照组、TGF-β组、TGF-β+Que-L（40 μmol/L）、TGF-β+Que-M（60 μmol/L）和TGF-β+Que-H（80 μmol/L）为药物治疗部分；各组阴性对照组、miR-146模拟剂（mimic）和抑制剂（inhibitor）为细胞转染部分。CCK-8法检测不同浓度的Que对细胞抑制情况，选出低、中、高3个剂量浓度用于后续细胞实验；细胞凋亡流式细胞术检测Que对HSCs细胞凋亡的影响；RT-qPCR法检测 HSC-T6细胞药物治疗以及细胞转染后miR-146、α-SMA、CollagenⅠ、TRAF6、PI3K和Akt mRNA表达情况；Western blot法检测以上各组中α-SMA、CollagenⅠ、TRAF6、PI3K、Akt和p-Akt蛋白表达情况；免疫荧光实验检测HSC-T6细胞药物治疗中α-SMA、CollagenⅠ蛋白表达强弱。结果 CCK-8实验结果显示，相同时间不同浓度时，与Control组相比，随着药物浓度的递增，细胞A450 nm值显著降低（P<0.05）；相同浓度不同时间比较，发现药物浓度在40、60、80 μmol/L时细胞A450 nm值间的差异最为显著。流式细胞术检测结果显示，Que各给药组总细胞凋亡率均显著升高（P<0.01）。Que对HSC-T6细胞的作用结果显示，与Control组相比，TGF-β组α-SMA、CollagenⅠ、TRAF6、PI3K和Akt的mRNA和蛋白表达水平显著升高（P<0.05）；与TGF-β相比，Que各治疗组α-SMA、CollagenⅠ、TRAF6、PI3K、Akt的mRNA和蛋白表达水平显著下调（P<0.05）。免疫荧光实验显示Que能显著减弱TGF-β组中α-SMA、CollagenⅠ蛋白的荧光强度。Que显著上调HSC-T6细胞中miR-146表达（P<0.01）；与各自阴性对照组相比，miR-146 mimic显著降低α-SMA、CollagenⅠ、TRAF6、PI3K、Akt的mRNA和蛋白表达水平（P<0.05），而miR-146 inhibitor使上述指标mRNA和蛋白表达水平显著逆转（P<0.05）。结论 Que通过上调miR-146影响PI3K/Akt信号通路来抑制HSCs增殖，促进其凋亡。

关键词: 槲皮素；肝星状细胞；miR-146；PI3K/Akt信号通路

Abstract: Objective To investigate whether quercetin induces apoptosis of hepatic stellate cells by regulating miR-146 to inhibit the PI3K/Akt signaling pathway. Methods Rat hepatic stellate cells (HSC-T6) were treated with TGF-β and different concentrations (40, 60 and 80 μmol/L) of quercetin, and the changes in cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry. RT-qPCR was used to detect the expression of miR-146 and mRNA expressions of α-SMA, collagenⅠ, TRAF6, PI3K and Akt in the treated cells, and the protein expressions of α-SMA, collagenⅠ, TRAF6, PI3K, Akt and p-Akt were detected using Western blotting. Immunofluorescence assay was used to detect the protein expression of α-SMA and collagenⅠ. The effects of transfection with miR-146 mimic and inhibitor on the protein expressions of the cells were also examined using Western blotting. Results Treatment with quercetin dose- and time-dependently inhibited the proliferation of HSC-T6 cells and significantly increased the total cell apoptosis rate (P<0.01). TGF-β-stimulated HSC-T6 cells showed significantly increased mRNA and protein expression levels of α-SMA, collagenⅠ, TRAF6, PI3K and Akt (P<0.05), which were significantly down-regulated by quercetin treatment (P<0.05). Quercetin significantly upregulated the expression of miR-146 in HSC-T6 cells (P<0.01), Transfection of the cells with miR-146 mimic significantly decreased the mRNA and protein expression levels of α-SMA, collagen Ⅰ, TRAF6, PI3K and Akt (P<0.05), and miR- 146 inhibitor produced the opposite effects (P<0.05). Conclusion Quercetin inhibits proliferation and promotes apoptosis of HSCs by upregulating miR-146 to inhibit the PI3K/Akt signaling pathway.

Key words: quercetin; hepatic stellate cells; miR-146; PI3K/Akt signaling pathway